Hepatitis C virus (HCV) establishes a chronic infection in a high percentage of infected individuals and is associated with progressive liver pathology, including cirrhosis and hepatocellular carcinoma. Antiviral drugs such as interferon alpha and ribavarin have had limited success in controlling HCV infection. As a result, it has become the leading cause for liver transplantation in the US. HCV is an enveloped virus containing a single stranded plus polarity RNA genome (˜9500 nt) which is infectious when injected directly into livers of chimpanzees. The 5′ untranslated region of viral RNA contains an internal ribosome entry site (IRES) which is used for translation of the single open reading frame into a large polyprotein. Viral structural and non-structural proteins are produced by proteolytic processing of the precursor. HCV has been a difficult virus to study due to the lack of an appropriate and reliable tissue culture system. The recent establishment of the HCV replicons that lack viral structural proteins has been a major advancement as it allows examination of viral gene expression and replication in tissue culture cells. However, the replicon based systems do not produce infectious virus as viral structural proteins are absent; consequently cell-to-cell spread of virus does not occur. Additionally, genetic analysis of the viral genome is hampered by the necessity of generating stable cell lines for the study of individual mutations.
The HCV polyprotein comprises, from the amino terminus to the carboxy terminus, the core protein (C), the envelope proteins (E1 and E2), and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). C codes for the core nucleocapsid protein, E1 and E2 are envelope proteins that coat the virus, NS2, NS3 and NS4A are involved in proteolytic processing of the HCV polyprotein, NS5B has RNA polymerase and RNA helicase activity. The functions of NS4A and NS5B are unknown.
Though the RNA genome of HCV has recently been cloned, there remains a need for a reliable tissue culture system for the generation of HCV. Using conventional methods, RNA viruses such as poliovirus, are produced in cells by transfecting cells using RNA. Transfecting cells using viral cDNA from RNA viruses, such as poliovirus has not been successful in producing such viruses. It was expected that HCV, an RNA virus, would also readily be produced by a transfecting process using RNA, however such a method was found not to be suitable for HCV. Further, it was expected that transfecting cells using viral cDNA would not be a successful method of producing HCV. However, the present invention presents a method of synthesizing infectious HCV by transfecting hepatocyte cells with a gene encoding HCV and then exposing uninfected cells to the HCV to form additional HCV. This synthesis method has been found to be successful.